Agarose immobilized enzymes

Another commonly used reaction for insolubilization of proteins and ligands through amino groups to polysaccharides is with 2,4,6-trichlorotriazine [18,19]. This procedure is extensively used in our laboratories in the preparation of agarose immobilized enzymes. 

Enzymes immobilized on PAN gel, unless stated otherwise. The actual phosphorylating agent, ATP, is regenerated either by acetyl phosphate and acetoldnase, or enolpyruvate phosphate and pyruvate kinase. Phosphorylations by a mixture of the common ribonucleotide triphosphates. Not isolated.4 Soluble enzymes. Enzymes immobilized on agarose.1 Enzymes enclosed in a dialysis bag. 

Unless otherwise stated, enzymes were immobilized on agarose. Immobilized in PAN gel Immobilized on silica gel-glutaialdehyde (a six-fold excess of CTP was utilized). 

Immobilized EEAC (Sigma Chemical Company) has been found equally effective and allows economical, large-scale hydrolyses of 2. The immobilized enzyme, which is covalently bonded to agarose beads, is easily recovered through centrifugation. Reuse does not compromise the enantioselectivity (unpublished results). 

Another fruitful area of research has been that of the sonochemical activation of immobilized enzymes where ultrasound appears to be particularly useful in increasing the transport of substrate to the enzyme. Using a-chymotrypsin (on agarose gel) and casein as substrate, a two-fold increase in activity was observed at 20 kHz [12]. Here the origin of the enhancement was thought to be associated with increased penetration of the casein into the support gel induced by cavitational effects close to the surface. However an increase in the activity of a-amylase (on porous polystyrene) was produced on irradiation with 7 MHz ultrasound [13]. This is a very significant result since at this high-frequency cavitation cannot occur and... 

The degradation of heparin by the reactor is a multistep process. Heparin and the heparin-antithrombin complex must first diffuse from the bulk phase to the surface of the immobilized enzyme particle. The two species diffuse into the agarose particles where they encounter immobilized heparinase. The heparin-anti thrombin complex is assumed to be sterically inhibited from binding to immobilized heparinase, and under these conditions only unbound heparin is enzymatically degraded. As unbound heparin is consumed, heparin dissociates from the heparin-antithrombin complex to generate more free heparin. The breakdown of heparin is given by the following chemical reaction ... 

To determine the effectiveness of the immobilized enzyme in blood, a column containing 5 g of agarose with the active enzyme was tested. Control columns contained the same amount of either untreated agarose or agarose containing denatured bilirubin oxidase. For the experiments in vivo, Gunn rats on lipid-free or regular diets were used. For the experiments in vitro, a blood reservoir of human umbilical-cord blood was used. 

Immobilization can be achieved by adsorption or covalent fixation of the biocatalyst to a solid support (e.g. surface-modified polymer or glass beads), by entrapment or by encapsulation in gel beads (e.g., agarose, polyacrylamide, alginate, etc.). Hundreds of immobilization methods have been described and reviewed in the literature [83-89], but only a limited set of methods has found real technical applications. The first large-scale applications of immobilized enzymes were established for the enantioseparation of D- and L-amino acids by Chib-ata, Tosa and co-workers at Tanabe Seiyaku Company. The Japanese achievements in the large-scale application of immobilized systems are very well documented in an excellent multi-author publication edited by Tanaka, Tosa and Kobayashi [90] (see also section 7). Some enzyme suppliers sell important industrial enzymes not only in the free form (solution or powder) but also immobilized on solid supports. 

In a previous work, Tardioli et al. [9] have immobilized CGTase from Thermoanaerobacter sp. by covalent attachment into glyoxy 1-agarose particles and obtained an activity recovery of about 32%, that is, five times the highest values obtained in this work (6.94% with sol-gel encapsulation). It is thought that with the latter method, in addition to the causes listed above for immobilized enzyme activity loss, the immobilization conditions and reagents used by the sol-gel method contribute to enzyme deactivation. 

The thermal stability of an immobilized enzyme is another important factor for the selection of an immobilization method. A method that shifts the maximum cataljlic activity for a region of higher temperatures is undoubtedly preferred. Tardioli et al. [9] obtained greater thermal stability for a Thermoanaerobacter sp. CGTase covalently bound to glyoxyl-agarose the temperature range shift for the maximum activity was from 80 to 85 (for the free enzyme) to just above 90 °C (for the immobilized enzyme). 

Rp protein immobilization yield (mg immobilizEd enzyme/mg contacted protein) Re enzyme immobilization yield (units of enzyme activity expressed in the biocatalyst/unit of enzyme activity contacted) SA specific activity of the biocatalyst (units of enzyme activity expressed/unit mass of biocatalyst). OIL octyl agarose immobilized lipase BIL butyl Sepabeads immobilized lipase... 

Investigation of the binding characteristics of urinary acid 3-D-gaIactosidase, 3-D-2-acetamido-2-deoxyglucosidase, a-D-galactosidase and a-L-fucosidase from patients with mucolipidosis II and mucolipidosis III to agarose-immobilized concanavalin A revealed a two to ten-fold decrease in the proportion of enzyme (that adsorbed on the lectin) activities from patients with mucolipidoses II and III. Neuraminidase treatment of the unabsorbed enzyme fraction did not... 

Recent references to the preparation of active immobilized forms of enzymes (for use as immobilized enzymes), of immunologically active macromolecules (for use as immunoadsorbents), and of various affinants (for use as affinity chromatographic matrices) using agarose cyclic imidocarbonate are summarized in the Tables which follow, the separate categories being the subject of... 

Table 1 Use of agarose cyclic imidocarbonates for the preparation of active immobilized enzymes...

Agarose cyclic imidocarbonate has been treated with polyethyleneimine and then modified with j3-l-[3,3-dimethyl-6-nitrospiro(indoline-2,2 -2/f-benzo-pyran)]propionic anhydride." Coupling of trypsin to the product yielded an active immobilized enzyme. 

A mixed disulphide derivative of agarose, agarose-glutathione-2(5-nitro-pyridyl)-disulphide, has been used to immobilize bovine galactosyltrans-ferase. As the immobilized enzyme retains biosynthetic activity, the thiol group of the enzyme that is involved in the coupling must be physically distinct... 

Reaction with agarose cyclic imido- Active immobilized enzyme for the 114 <3- s... 

Unspecified reaction with agarose, dextran, aminoethyl-cellulose, and several inorganic carriers Active immobilized enzyme 741 o c ... 

CMP-acylneuraminate synthase Reaction with agarose cyclic imido-carbonate Active immobilized enzyme synthesis of CMP-A-acetylneuraminic and CMP-A-glycoloylneuraminic acids 122... 

Cross-linking onto agarose using ethylene Active immobilized enzyme assessment of 807 ... 

The immobilized enzyme was used for repeated synthesis of the above sialylated trisaccharides without adding detergent to the system, which was used in previous synthesis with the soluble enzyme (42), The use of detergent complicates isolation of the products. The stability of the enzyme preparation was good and no decrease of activity was observed on repeated preparative synthesis. Recently, immobilization to CNBr-activated agarose of the a2-6sialyltransferase was reported (50),... 

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